Cellular protein degradation is a regulated process involved in the control of a broad array of biological processes. The ubiquitin-proteasome cascade is the major system for proteolysis of cytosolic proteins in eukaryotic cells. Tripeptidyl peptidase II (TPP II), a 6 MDa subtilisin-like serine peptidase, cleaves proteasome products to produce MHC class I antigenic peptides and short peptides that can be used as substrates by other exo-peptidases. TPP II mediated endo-peptidolytic activity generates the human immunodeficiency virus (HIV) epitope, Nef. TPP II also appears to be a peptidase critical for cell survival in cases where proteasome activity is sub-optimal, such as has been observed in apoptosis-resistant tumor cells. The TPP II holo-complex is a spindle-shaped structure composed of two twisted strands, each containing 10 dimers;active sites are formed through the sequential association of dimers. As two dimers come into contact along a growing strand, one monomer from each dimer becomes activated. The long-range objective of this research proposal is to understand the molecular mechanism of TPP II mediated peptidolysis, complex assembly, and assembly-dependent activation. In support of achieving this objective we will determine, by x-ray crystallographic methods, the structure of the 600 kDa TPP II tetramer, which has been found to retain full enzymatic activity in two of its four subunits. A high-resolution structure of the tetramer will provide structural details of the two active and two inactive subunits which, in turn, will yield insight into the process of assembly-dependent activation. This work will also include the structure determination of the tetramer with inhibitors bound. We also propose to obtain a density map of the 6 MDa complex to a resolution better than 1.5 nm using single particle cryo-EM methods. We will use this higher resolution EM-based map and the atomic structure of the TPP II tetramer to construct a high-resolution model of the 6 MDa holo-complex. The structural information gained from the atomic model of the tetramers together with a high resolution model of the 6 MDa holo-complex will provide molecular level insight into TPP II substrate access and binding, the peptidolytic process, and the mechanisms involved in regulating assembly of the complex to the length consistently observed in vivo. This knowledge, in turn, may support the design of future therapeutic strategies for the treatment of certain cancers and HIV infection.